Epigenetic processes are key driving forces in carcinogenesis and are modulated by essential nutrients in the diet. In particular, zinc deficiency (ZD) reduces the utilization of methyl groups from S-adenosylmethionine (SAM) in rat liver, resulting in global DNA hypomethylation. The impact of ZD on DNA methylation in carcinogenesis, however, is largely unexplored. Epidemiological studies have shown that dietary ZD is associated with cancer of the upper aerodigestive tract (UADT), including esophageal and oral cancers. We have developed in vivo cancer models that reproduce features of human UADT cancer. In rodents, ZD alone creates a precancerous condition in the UADT by causing cell proliferation and extensive changes in gene expression, thereby enhancing carcinogenesis. Zinc replenishment (ZR) rapidly reverses cell proliferation, corrects abnormal gene expression, and inhibits tumorigenesis. Importantly, p53+/- mice on a ZD diet show speedy development and progression of UADT cancer by low doses of carcinogens. Our new data showed overexpression of DNA methyltransferase enzymes DNMT3A and DNMT1 in ZD versus ZS rat esophagus and their repression upon ZR. Based on these observations, we hypothesized: A) low zinc intake increases the risk of UADT cancer that ZR reduces through modulation of epigenetic processes;and B) combined deficiency of zinc and p53 in ZD:p53+/- mice induces specific methylation patterns that favor UADT tumor progression, which are different from those displayed in ZS:p53+/-, ZD:p53+/+ or ZS:p53+/+ mice. Aim 1. Use the in vivo ZD and ZR rat esophagus system to determine: A) whether DNA methylation is a mechanism that underlies the phenotypic and genetic changes effected by dietary zinc, by determining in precancerous ZD esophagus, normal zinc-sufficient (ZS) esophagus, and restored ZR esophagus: 1) intracellular levels of SAM, S- adenosylhomocysteine (SAH), activity of Dnmt enzymes, and global DNA methylation status, and 2) methylation status of CpG islands in regulatory regions of specific genes, including the tumor suppressor genes CDKN2A and FHIT, the repair gene MGMT, and the stress-inducible gene MT-1;and B) if the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine can correct ZD-modulated epigenetic processes and inhibit chemically induced esophageal carcinogenesis in ZD rats. Aim 2. To study genome-wide promoter methylation in esophagus and tongue in DNA from ZD:p53+/-, ZS:p53+/-, ZD:p53+/+, and ZS:p53+/+ mice during UADT carcinogenesis, by using restriction landmark genomic scanning (RLGS) method and the mouse NotI-EcoRV arrayed library. This AIM will determine whether linkages can be established between DNA methylation patterns and the diverse tumorigenic outcomes in p53+/- and p53+/- mice on ZD/ZS diets. The results of these studies will provide a new understanding of how dietary zinc impacts epigenetic processes in UADT cancer development and prevention. In addition, the results will identify targets for chemoprevention, diagnosis, and treatment of this deadly cancer.